9.
GENERAL REFERENCE
325
is often necessary to search diligently for a preparation which shows
detail adequately.
Electron images are not solely the result of electron scattering. Diffraction effects are also important in their interpretation. These, in turn,
are quite dependent on focal levels as Fig. 40 demonstrates. Diffraction
is minimal at true focus [Fig. 40(b)]. It is at this level that contrast also
is minimal, for diffraction phenomena ordinarily enhance contrast [Fig.
40(c) and (d)], albeit at the expense of resolution. In particular, the additive effect of diffraction at slightly below the true focal plane [Fig. 40(c)]
often results in micrographs of optimal quality in terms of visualization
of structure at low and moderate levels of resolution. But the images are
not as meaningful as true focus images at high magnification.
Diffraction effects which result from overfocusing are more complicated
as Fig. 40(a) demonstrates. Structures that are truly punctate may appear
to have hollow centers, and dense rims. Patterned structures such as
myelin may have density differences obliterated as in this instance in
which the interperiod band became indistinguishable from the principal
period line. Thus it is evident that under some circumstances investigators
can be seriously misled in their interpretations by overfocused diffraction
patterns unless they are forewarned and on guard.
GENERAL REFERENCE
Lester, H. M., Photo-Lab Index, Morgan and Lester, New York. (A loose-leaf cumulative
formulary of photographic materials and procedures. Quarterly supplements are
issued to keep it up to date. New editions are issued almost yearly. This is the
standard "bible" of the American professional photographer, but European
materials are also included with considerable detail. It includes an extensive
bibliography relative to photographic processes.)
FIG. 40. A through-focus series of micrographs of myelin of cat spinal cord. The
area of the section contained a small hole which is printed much more darkly than
the rest so that diffraction patterns (Fresnel fringes) are evident at its edge, and thus
demonstrate focal level (Chapter 9.3, Fig. 34).
(b) Nearly true focus. Specimen contrast is minimal, resolution maximal.
(c) and (d) Below-focus. Diffraction exaggerates contrast, but at the expense of resolution. Nevertheless a slightly below-focus micrograph often is most informative.
(a) Over-focus. Diffraction exaggerates contrast in a complex way so that the interperiod band of the myelin shows the same contrast as the principal period. What are
truly punctate spots now appear as having hollow centers and dark halos.
Tissue fixed first by perfusion of buffered formaldehyde, and then treated with
osmium tetroxide. Embedded in Epon. Double stained, first with uranium, and then
lead.
GENERAL REFERENCE
325
is often necessary to search diligently for a preparation which shows
detail adequately.
Electron images are not solely the result of electron scattering. Diffraction effects are also important in their interpretation. These, in turn,
are quite dependent on focal levels as Fig. 40 demonstrates. Diffraction
is minimal at true focus [Fig. 40(b)]. It is at this level that contrast also
is minimal, for diffraction phenomena ordinarily enhance contrast [Fig.
40(c) and (d)], albeit at the expense of resolution. In particular, the additive effect of diffraction at slightly below the true focal plane [Fig. 40(c)]
often results in micrographs of optimal quality in terms of visualization
of structure at low and moderate levels of resolution. But the images are
not as meaningful as true focus images at high magnification.
Diffraction effects which result from overfocusing are more complicated
as Fig. 40(a) demonstrates. Structures that are truly punctate may appear
to have hollow centers, and dense rims. Patterned structures such as
myelin may have density differences obliterated as in this instance in
which the interperiod band became indistinguishable from the principal
period line. Thus it is evident that under some circumstances investigators
can be seriously misled in their interpretations by overfocused diffraction
patterns unless they are forewarned and on guard.
GENERAL REFERENCE
Lester, H. M., Photo-Lab Index, Morgan and Lester, New York. (A loose-leaf cumulative
formulary of photographic materials and procedures. Quarterly supplements are
issued to keep it up to date. New editions are issued almost yearly. This is the
standard "bible" of the American professional photographer, but European
materials are also included with considerable detail. It includes an extensive
bibliography relative to photographic processes.)
FIG. 40. A through-focus series of micrographs of myelin of cat spinal cord. The
area of the section contained a small hole which is printed much more darkly than
the rest so that diffraction patterns (Fresnel fringes) are evident at its edge, and thus
demonstrate focal level (Chapter 9.3, Fig. 34).
(b) Nearly true focus. Specimen contrast is minimal, resolution maximal.
(c) and (d) Below-focus. Diffraction exaggerates contrast, but at the expense of resolution. Nevertheless a slightly below-focus micrograph often is most informative.
(a) Over-focus. Diffraction exaggerates contrast in a complex way so that the interperiod band of the myelin shows the same contrast as the principal period. What are
truly punctate spots now appear as having hollow centers and dark halos.
Tissue fixed first by perfusion of buffered formaldehyde, and then treated with
osmium tetroxide. Embedded in Epon. Double stained, first with uranium, and then
lead.